Abstract:

The invention features a method for determining methyl transferase
activity of a polypeptide and screening for modulators of methyl
transferase activity. The invention further provides a method or
pharmaceutical composition for prevention or treating of colorectal
cancer or hepatocellular carcinoma using the modulator.

Claims:

1. A method of measuring methyl transferase activity of a polypeptide,
said method comprising the steps of:a. contacting a polypeptide selected
from the group consisting of:i. a polypeptide comprising the amino acid
sequence of SEQ ID NO: 51 (ZNFN3A1);ii. a polypeptide comprising the
amino acid sequence of SEQ ID NO: 51 wherein one or more amino acids are
substituted, deleted, or inserted, and said polypeptide has a biological
activity equivalent to the polypeptide consisting of the amino acid
sequence of SEQ ID NO: 51;iii. a polypeptide that comprises the amino
acid sequence having at least about 80% homology to SEQ ID NO: 51; andiv.
a polypeptide encoded by a polynucleotide that hybridizes under stringent
conditions to a polynucleotide consisting of the nucleotide sequence of
SEQ ID NO: 50, wherein the polypeptide has a biological activity
equivalent to a polypeptide consisting of the amino acid sequence of SEQ
ID NO: 51;with a substrate to be methylated and a cofactor under the
condition capable of methylation of the substrate;b. detecting the
methylation level of the substrate; andc. measuring the methyl
transferase activity by correlating the methylation level of step (b)
with the methyl transferase activity.

2. The method of claim 1, wherein the substrate is a histone or the
fragment thereof comprising an at least methylation region.

3. The method of claim 1, wherein the methylation region is a histone H3
lysine 4.

4. The method of claim 1, wherein the cofactor is a
S-adenosyl-L-methionine.

5. The method of claim 1, wherein the condition capable of methylation of
the substrate is provided in the existence of heat shock protein 90A
(HSP90A).

6. The method of claim 1, wherein the polypeptide is contacted with the
substrate and cofactor in the presence of an enhancing agent for the
methylation.

7. The method of claim 6, wherein the enhancing agent for the methylation
is S-adenosyl homocysteine hydrolase (SAHH).

8. A method identifying an agent that modulate methyl transferase
activity, said method comprising the steps of:a. contacting a polypeptide
selected from the group consisting of:i. a polypeptide comprising the
amino acid sequence of SEQ ID NO: 51;ii. a polypeptide that comprises the
amino acid sequence of SEQ ID NO: 51 wherein one or more amino acids are
substituted, deleted, or inserted, and said polypeptide has a biological
activity equivalent to the polypeptide consisting of the amino acid
sequence of SEQ ID NO: 51;iii. a polypeptide that comprises the amino
acid sequence having at least about 80% homology to SEQ ID NO: 51; andiv.
a polypeptide encoded by a polynucleotide that hybridizes under stringent
conditions to a polynucleotide consisting of the nucleotide sequence of
SEQ ID NO: 50, wherein the polypeptide has a biological activity
equivalent to a polypeptide consisting of the amino acid sequence of SEQ
ID NO: 51;with a substrate to be methylated and a cofactor in the
presence of the test compound under the condition capable of methylation
of the substrate;b. detecting the methylation level of the substrate;
andc. comparing the methylation level to a control levelwherein an
increase or decrease in the methylation level compared to control level
indicates that the test compound modulates methyl transferase activity.

9. A kit for detecting for an activity of a test compound to regulate
methyl transferase activity, said kit comprising the components of:a. a
polypeptide selected from the group consisting of:i. a polypeptide
comprising the amino acid sequence of SEQ ID NO: 51;ii. a polypeptide
comprising the amino acid sequence of SEQ ID NO: 51 wherein one or more
amino acids are substituted, deleted, or inserted and said polypeptide
has a biological activity equivalent to the polypeptide consisting of the
amino acid sequence of SEQ ID NO: 51;iii. a polypeptide that comprises
the amino acid sequence having at least about 80% homology to SEQ ID NO:
51; andiv. a polypeptide encoded by a polynucleotide that hybridizes
under stringent conditions to a polynucleotide consisting of the
nucleotide sequence of SEQ ID NO: 50, wherein the polypeptide has a
biological activity equivalent to a polypeptide consisting of the amino
acid sequence of SEQ ID NO: 51;b. a substrate capable of methylation by
the polypeptide of (a),c. a cofactor for the methylation of the
substrate, andd. HSP90A.

10. The kit of claim 9, wherein the substrate is a histone or the fragment
thereof comprising an at least methylation region.

12. A method of screening for a compound for treating colorectal cancer or
hepatocellular carcinoma, said method comprising the steps of:a.
identifying the compound having an activity to modulate methyl
transferase activity by the method of claim 7, andb. selecting a compound
that decrease the methylation level of the substrate compared to a
control level.

13. A method of screening for a compound for treating colorectal cancer or
hepatocellular carcinoma, said method comprising the steps of:a.
contacting a polypeptide selected from the group consisting of:i. a
polypeptide comprising the amino acid sequence of SEQ ID NO: 51;ii. a
polypeptide comprising the amino acid sequence of SEQ ID NO: 51 wherein
one or more amino acids are substituted, deleted, or inserted and said
polypeptide has a biological activity equivalent to the polypeptide
consisting of the amino acid sequence of SEQ ID NO: 51;iii. a polypeptide
that comprises the amino acid sequence having at least about 80% homology
to SEQ ID NO: 51; andiv. a polypeptide encoded by a polynucleotide that
hybridizes under stringent conditions to a polynucleotide consisting of
the nucleotide sequence of SEQ ID NO: 50, wherein the polypeptide has a
biological activity equivalent to a polypeptide consisting of the amino
acid sequence of SEQ ID NO: 51;with a heat shock protein 90A polypepetide
(HSP90A) in the presence of a test compound;b. detecting binding between
the polypeptide and HSP90A;c. comparing the binding of the polypeptide
and HSP90A in the presence of the test compound with that in the absence
of the test compound, andd. selecting a test compound which decreases the
binding of the polypeptide and HSP90A.

14. A kit for screening for a compound for treating colorectal cancer or
hepatocellular carcinoma, said kit comprising the components of:a. a
polypeptide selected from the group consisting of:i. a polypeptide
comprising the amino acid sequence of SEQ ID NO: 51;ii. a polypeptide
comprising the amino acid sequence of SEQ ID NO: 51 wherein one or more
amino acids are substituted, deleted, or inserted and said polypeptide
has a biological activity equivalent to the polypeptide consisting of the
amino acid sequence of SEQ ID NO: 51;iii. a polypeptide that comprises
the amino acid sequence having at least about 80% homology to SEQ ID NO:
51; andiv. a polypeptide encoded by a polynucleotide that hybridizes
under stringent conditions to a polynucleotide consisting of the
nucleotide sequence of SEQ ID NO: 50, wherein the polypeptide has a
biological activity equivalent to a polypeptide consisting of the amino
acid sequence of SEQ ID NO: 51;with a heat shock protein 90A polypepetide
(HSP90A) in the presence of a test compound; andb. HSP90A.

15. A method of screening for a compound for treating colorectal cancer or
hepatocellular carcinoma, said method comprising the steps of:a.
contacting a polypeptide comprising an contiguous amino acid sequence
that selected from the amino acid sequence of SEQ ID NO: 51, and wherein
the amino acid sequence comprises either or both of NHSCDPN (SEQ ID
NO:52) and GEELTICY (SEQ ID NO:53), with an S-adenosyl-L-methionine in
the presence of a test compound;b. detecting binding between the
polypeptide and S-adenosyl-L-methionine;c. comparing the binding of the
polypeptide and S-adenosyl-L-methionine in the presence of the test
compound with that in the absence of the test compound, andd. selecting a
test compound which decreases the binding of the polypeptide and
S-adenosyl-L-methionine.

16. A kit for screening for a compound for treating colorectal cancer or
hepatocellular carcinoma, said kit comprising the components of:a. a
polypeptide comprising an contiguous amino acid sequence that selected
from the amino acid sequence of SEQ ID NO: 51, and wherein the amino acid
sequence comprises either or both of NHSCDPN (SEQ ID NO:52) and GEELTICY
(SEQ ID NO:53); andb. S-adenosyl-L-methionine.

17. A composition for alleviating a symptom of colorectal cancer or
hepatocellular carcinoma, said composition comprising a pharmaceutically
effective amount of a compound that decreases ZNFN3A1-mediated
methylation and a pharmaceutically acceptable carrier.

18. A method for alleviating a symptom of colorectal cancer or
hepatocellular carcinoma comprising contacting a colorectal cancer cell
or a heptocellular carcinoma cell with a pharmaceutically effective
amount of a compound that decreases ZNFN3A1-mediated methylation.

19. A method for alleviating a symptom of colorectal cancer or
hepatocellular carcinoma comprising contacting a colorectal cancer cell
or a heptocellular carcinoma cell with a pharmaceutically effective
amount of a compound that decreases an interaction between ZNFN3A1 and
HSP90A.

20. A method for alleviating a symptom of colorectal cancer or
hepatocellular carcinoma comprising contacting a colorectal cancer cell
or a heptocellular carcinoma cell with a pharmaceutically effective
amount of a compound that decreases an interaction between ZNFN3A1 and
S-adenosyl-L-methionine.

Description:

[0001]This application claims the benefit of U.S. Provisional Application
Ser. No. 60/538,658 filed Jan. 23, 2004, the contents of which are hereby
incorporated by reference in its entirety.

TECHNICAL FIELD

[0002]The present invention relates to transcriptional regulation.

BACKGROUND ART

[0003]Hepatocellular carcinoma (HCC) is one of the most common cancers
worldwide and its incidence is gradually increasing in Japan as well as
in United States (Akriviadis E A, et al., Br J. Surg. 1998 October;
85(10):1319-31). Although recent medical advances have made great
progress in diagnosis, a large number of patients with HCCs are still
diagnosed at advanced stages and their complete cures from the disease
remain difficult. In addition, patients with hepatic cirrhosis or chronic
hepatitis have a high risk to HCCs, they may develop multiple liver
tumors, or new tumors even after complete removal of initial tumors.
Therefore development of highly effective chemotherapeutic drugs and
preventive strategies are matters of pressing concern.

[0004]Colorectal carcinoma is a leading cause of cancer deaths in
developed countries. Specifically, more than 130,000 new cases of
colorectal cancer in the United States are reported each year. Colorectal
cancer represents about 15% of all cancers. Of these, approximately 5%
are directly related to inherited genetic defects. Many patients have a
diagnosis of pre-cancerous colon or rectal polyps before the onset of
cancer. While many small colorectal polyps are benign, some types may
progress to cancer. The most widely used screening test for colorectal
cancer is colonoscopy. This method is used to visualize a suspicious
growth and/or take a tissue biopsy. Typically, the tissue biopsy is
histologically examined and a diagnosis delivered based on the
microscopic appearance of the biopsied cells. However, this method is
limited in that it yields subjective results and can not be used for very
early detection of pre-cancerous states. The development of a sensitive,
specific and convenient diagnostic system for detecting very early-stage
colorectal cancers or pre-malignant lesions is highly desirable as it
could ultimately eliminate this disease.

SUMMARY OF THE INVENTION

[0005]The present invention is based in part on the discovery of the
methyl transferase activity of ZNFN3A1, a polypeptide which is involved
in proliferation of cancer cells. Moreover, the methyl transferase
activity of ZNFN3A1 is expressed in the presence of 90-kD heat shock
protein (HSP90A).

[0006]Accordingly, the invention features a method of measuring methyl
transferase activity by contacting a polypeptide of the invention with a
methyl transferase substrate and a co-factor under conditions suitable
for methylation of the substrate and detecting the methylation level of
the substrate. The polypeptide of the invention is a ZNFN3 μl
polypeptide or functional equivalent thereof. For example, a polypeptide
of the invention may comprise the amino acid sequence of SEQ ID NO: 51.
Alternatively, the polypeptides of the invention can include an amino
acid sequence of SEQ ID NO: 51 where one or more amino acids are
substituted, deleted, or inserted and the polypeptide has a biological
activity of the polypeptide of SEQ ID NO:51. Biological activity of the
polypeptide of SEQ ID No:51 includes for example the promotion of cell
proliferation and the transcriptional activation of target genes.
Additionally, the polypeptide includes a 428-amino acid protein encoded
by the open reading frame of SEQ ID NO:50 or a polynucleotide that
hybridizes under stringent conditions, e.g., low or high, to the
nucleotide sequence of SEQ ID NO:50 and has a biological activity of SEQ
ID NO:51. A low stringent condition is, for example, 42° C.,
2×SSC, 0.1% SDS, or preferably 50° C., 2×SSC, 0.1%
SDS. Preferably, a high stringent conditions is used. A high stringent
condition is, for example, washing 3 times in 2×SSC, 0.01% SDS at
room temperature for 20 min, then washing 3 times in 1×SSC, 0.1%,
SDS at 37° C. for 20 min, and washing twice in 1×SSC, 0.1%
SDS at 50° C. for 20 min. However, several factors such as
temperature and salt concentration can influence the stringency of
hybridization and one skilled in the art can suitably select the factors
to achieved the requisite stringency. Optionally, the polypeptide is
further contacted with 90-kD heat shock protein. Methylation is defined
as the catalysis of the transfer of a methyl group to an another
compound, e.g., acceptor molecule. Methylation is detected by methods
such as using a radioactive methyl donor. The substrate is any compound
capable of accepting a methyl group such as a protein, a nucleic acid
(RNA or DNA) or a small molecule. For example, the substrate is a histone
or a fragment of a histone containing the methylation region. Actually,
it is confirmed that histone H3 lysine 4 can be methylated by ZNFN3
μl. Therefore, histone H3, or the fragment thereof containing lysine
4, is useful as the substrate. The co-factor, e.g., the methyl donor, is
any compound capable of donating a methyl group. For example, the
co-factor is S-adenosyl-L-methionine. Suitable conditions for methylation
include for example basic buffer conditions know in the art such as
Tris-HCl.

[0007]The invention further provides methods of identifying an agent that
modulates (e.g., increases or decreases) methyl transferase activity by
contacting a polypeptide of the invention with a methyl transferase
substrate and a co-factor under conditions suitable for methylation of
the substrate in the presence of a test agent and determining the
methylation level of the substrate. A decrease of the level of
methylation compared to a normal control methylation level indicates that
the test agent is an inhibitor of methyl transferase activity. Compounds
that inhibit (e.g., decreases) methyl transferase activity are useful for
treating, preventing or alleviating a symptom of colorectal cancer or
heptaocellualar carcinoma. For example, the compounds inhibit the
proliferation of cancer cells. Alternatively, an increase of the level or
activity compared to a normal control level indicates that the test agent
is an enhancer of methyl transferase activity. By normal control level is
meant a level of methylation of a substrate detected in the absence of
the test compound.

[0008]The invention provides a method for screening a compound for
treating colorectal cancer or hepatocellular carcinoma by contacting a
polypeptide with a heat, shock protein 90A (HSP90A) polypeptide in the
presence of a test agent and detecting binding between the polypeptide
and HSP90A. The binding of the polypeptide and HSP90A in the presence of
the test compound compared to the absence of the test compound. Test
compounds which decrease the binding of the polypeptide and HSP90A are
selected. Binding of the polypeptide and HSP90A is defined as a
non-covalent association between the polypeptide and HSP90A. Binding is
measures by methods known in the art such as a yeast two-hybrid screening
system.

[0009]The invention also encompasses compositions and methods for
alleviating a symptom of a colorectal cancer or hepatocellular carcinoma
by contacting a colorectal cancer cell or hepatocellular carcinoma cells
with a compound identified as described above. For example, a method of
treating a either or both of colorectal cancer and hepatocellular
carcinoma is carried out by administering to a mammal, e.g. a human
patient having been diagnosed with such a disease state, with a
composition containing a pharmaceutically effective amount of the
compound identified as described above and a pharmaceutical carrier.

[0010]The invention also provides a kit for detecting methyl transferase
activity of a compound with a methyl transferase polypeptide, a
substrate, a cofactor, and HSP90A. The reagents are packaged together in
the form of a kit. The reagents are packaged in separate containers,
e.g., a methyl transferase polypeptide of the invention, substrate,
co-factor, a control reagent (positive and/or negative), and/or a
detectable label. Another embodiment of the invention is a kit for
detecting the activity of a test compound to regulate the
methyltransferase activity, and/or binding between ZNFN3A1 polypeptide of
the invention and heat shock protein 90 A polypeptide (HSP90A). The kit
includes ZNFN3A1 polypeptide or fragment thereof and an HSP90A
polypeptide. In some aspects of the embodiment ZNFN3A 1 is a polypeptide,
preferably a recombinant polypeptide, comprising an amino acid sequence
having a SET domain of native ZNFN3A1. Furthermore, the invention also
provides a kit for screening for a compound for treating colorectal
cancer or hepatocellular carcinoma, said kit comprising the components of
a polypeptide comprising an contiguous amino acid sequence that selected
from the amino acid sequence of SEQ ID NO: 51, and wherein the amino acid
sequence comprises either or both of NHSCDPN (SEQ ID NO:52) and GEELTICY
(SEQ ID NO:53); and S-adenosyl-L-methyonine. Instructions (e.g., written,
tape, VCR, CD-ROM, etc.) for carrying out the assay are included in the
kit. The assay format of the kit is a transferase assay or binding assay
known in the art.

[0011]Unless otherwise defined, all technical and scientific terms used
herein have the same meaning as commonly understood by one of ordinary
skill in the art to which this invention belongs. Although methods and
materials similar or equivalent to those described herein can be used in
the practice or testing of the present invention, suitable methods and
materials are described below. All publications, patent applications,
patents, and other references mentioned herein are incorporated by
reference in their entirety. In case of conflict, the present
specification, including definitions, will control. In addition, the
materials, methods, and examples are illustrative only and not intended
to be limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

[0012]FIG. 1A is an illustration depicting conserved sequences in the SET
domains of methyl transferases.

[0013]FIG. 1B is a photograph of a SDS-PAGE gel showing the interaction
between wild-type ZNFN3A1 and S-adenosyl-L-methyonine (SAM). Equal amount
of wild-type or mutant ZNFN3A1 (arrowhead) was incubated with
[3H]-labeled SAM (top panel). SAM-associated ZNFN3A1 (arrowhead) was
detected by fluorogram (bottom panel).

[0014]FIG. 1C is a photograph of a In vitro HMTase assay of SET-domain of
ZNFN3 μl with/without recombinant HSP90A. SET7 served as a control.

[0015]FIG. 2A is a photograph of a in vitro histone H3-K4
methyltransferasease assay. Histone H3 was incubated with wild-type or
mutant ZNFN3 μl, or SET7 in the presence SAM and HSP90A.

[0016]FIG. 2B is a photograph of showing inhibition of H3-K4
di-methylation by the addition of specific peptides to di-methylated
H3-K4.

[0017]FIG. 2C is a photograph of a in vitro histone H3-K9
methyltransferasease assay. SUV39H1 served as a positive control.

[0021]FIG. 3D is a bar chart showing the effect of SAHH on the HMTase
activity of ZNFN3A1.

[0022]FIG. 4A is a bar chart showing the effect of oncogenic activity of
ZNFN3A1 in HEK293 cells. Number of viable cells was measured by Cell
Counting Kit-8 at Day14 after the transfection. *, a significant
difference (p<0.05) determined by a Fisher's protected
least-significant test.

[0023]FIG. 4B is a bar chart showing the effect of oncogenic activity of
ZNFN3A1 in HCT116 cells. Number of viable cells was measured by Cell
Counting Kit-8 at Day14 after the transfection. *, a significant
difference (p<0.05) determined by a Fisher's protected
least-significant test.

[0025]FIG. 5B is a bar chart showing the cell viability of hepatoma cell
lines were measured by Cell Counting Kit-8 14 days after transfection of
plasmids. Cells were selected with 1.0 μg/μl G418-containing DMEM
for HepG2, 0.8 μg/μl G418-containing DMEM for Huh7 and Alexander.
*, a significant difference (p<0.05) determined by a Fisher's
protected least-significant test.

[0026]FIG. 6A is a photograph of a showing exogeneous expression of
ZNFN3A1 in HEK293 cells. Time-dependent expression of ZNFN3A1 in cells
transfected with pcDNA (Mock) or pcDNA-ZNFN3A1 was examined by western
blot analysis.

[0027]FIG. 6B is a photograph of a showing expression of candidate
downstream genes in response to exogenous ZNFN3A1. Semi-quantitative
RT-PCR analysis was performed using RNA from HEK293 cells transfected
with pcDNA-ZNFN3A1 or mock.

[0028]FIG. 7A is an illustration depicting the putative ZNFN3A1-binding
sequences in the 5'-flanking region of Nkx2.8. The HSP90A-dependent
transactivation of Nkx2.8 by ZNFN3A1 express through its interaction with
putative binding sequences.

[0029]FIG. 7B is a photograph of a depicting identification of
ZNFN3A1-binding elements in the Nkx2.8 promoter region by ChIP assay.

[0030]FIG. 7C is a bar chart depicting the results of an in vitro binding
assay between recombinant GST-ZNFN3A1 and a double-stranded DNA probe
containing the ZNFN3A1-binding element (ZBE).

[0032]FIG. 7E is a photograph of a gel showing expression of Nkx2.8 in
response to exogeneous expression of wild-type (lane 2 and 3) or mutant
(lane 4 and 5) ZNFN3A1 in HEK293 cells. Addition of HSP90A-specific
inhibitor, geldanamycin, diminished the enhanced expression of Nkx2.8
caused by wild-type ZNFN3A1 (lane 3).

[0033]FIG. 7F is a bar chart showing the effect of wild-type or mutant
ZNFN3A1 on the luciferase activity in HEK293-Nkx2.8Luc cells that contain
integrated Nkx2.8 promoter-luciferase gene in the genome.

[0034]FIG. 8A is a photograph of a showing interaction between endogeneous
ZNFN3A1 and the ChIP-4 region of Nkx2.8 in hepatoma cells.

[0035]FIG. 8B is a photograph of a showing interaction between
di-methylated histone H3 lysine 4 (H3-K4) and the ChIP-4 in the presence
of ZNFN3A1 in HEK293 cells.

DETAILED DESCRIPTION OF THE INVENTION

[0036]The present invention is based in part on the discovery of a novel
histone methyl transferase, ZNFN3A1, which is involved in proliferation
of cancer cells. The histone methyl transferase activity of ZNFN3A1 is
expressed in the presence of 90-kD heat shock protein (HSP90A), thus
HSP90A plays a role in for a histone methyl transferase activity.

[0037]ZNFN3A1 expression is markedly elevated in colorectal carcinoma and
hepatocellular carcinome (HCCs) compared to non-cancerous liver and
colorectal tissues (WO 03/27143). The ZNFN3A1 cDNA consists of 1622
nucleotides that contain an open reading frame of 1284 nucleotides as set
forth in SEQ. ID. NO.:50. The open reading frame encodes a 428-amino acid
protein with a zinc finger motif and a SET domain, as shown in SEQ. ID.
NO.:51. The subcellular localization of ZNFN3A1 protein is altered during
cell cycle progression and by the density of cultured cells. ZNFN3A1
protein accumulates in the nucleus when cells are in middle to late S
phase or cultured in sparse conditions. Whereas, ZNFN3A1 protein
localizes in the cytoplasm as well as in the nucleus when cells are in
other phases of the cell cycle or grown in a dense condition.

[0038]ZNFN3A1 contains a SET domain defined by two conserved amino acid
sequences, "NHSCXXN" (SEQ ID NO:54) and "GEELXXXY" (SEQ ID NO:55). (FIG.
1A) Genes which encode proteins with a SET domain(s) are classified into
four families, namely SUV39, SET1, SET2 and RIZ families according to the
homology of their SET domains. The SET domain of ZNFN3A1 does not contain
any pre-SET, post-SET, AWS, SANT or C2H2 domains, which are conserved in
these subfamilies, thus ZNFN3A1 may constitute a new class of subfamily
of SET domain proteins.

[0039]ZNFN3A1 directly associates with a RNA helicase KIAA0054, and forms
a complex with RNA polymerase II, which activates transcription of
downstream genes including epidermal growth factor receptor (EGFR)
through a direct binding of the complex with an element of "(C)CCCTCC(T)"
in the 5' flanking region of the EGFR gene. Moreover, ZNFN3A1 has been
shown to associate with RNA helicase (HELZ) and 90-kD heat shock protein
(HSP90A).

[0040]Exogenous expression of ZNFN3A1 into NIH3T3 cells resulted in
increased cell growth. In contrast, suppression of its expression with
antisense S-oligonucleotides resulted in a significant growth-inhibition
of hepatoma cells. Furthermore, it was confirmed that siRNA of ZNFN3A1
can also inhibit the proliferation of hepatoma cells and corolectal
adenocarcinomas (WO2004/76623). These findings indicate that ZNFN3A1
renders oncogenic activities to cancer cells by transcriptional
activation of target genes including EGFR through a complex with RNA
helicase and RNA polymerase II, and that inhibition of the activity of
the complex is a strategy for the treatment of colorectal carcinoma and
hepatocellular carcinoma. Deregulation of other SET domain proteins have
been shown to be involved in human neoplasms. For example in human
leukemia, frequent translocations are observed in MLL (10,11), the human
homolog of Drosophila trithorax gene that belongs to SET1 family.
Although it is unclear whether loss or gain of MLL function is
responsible for the oncogenesis, MLL activates transcription of the Hox
gene through H3 lysine 4-specific methylation mediated by methylase
activity of the SET domain (12) through its direct binding to the Hox
promoter sequences. MLL2 and EZH2, two members of SET1 family are
amplified in pancreatic cancers, gliomas, or hormone-refractory,
metastatic prostate cancers (13-15).

[0041]The invention thus provides a method of screening for a compound
that modulates ZNFN3A1 methyltransferase activity. The method is
practiced by contacting a ZNFN3A1 polypeptide or functional equivalent
thereof having methyl transferase activity with one or more candidate
compounds, and assaying methyl transferase activity of the contacted
ZNFN3A1 or the functional equivalent. A compound that modulates methyl
transferase activity of the ZNFN3A1 or functional equivalent is thereby
identified.

[0042]In the present invention, the term "functionally equivalent" means
that the subject protein has the same or substantially the same methyl
transferase activity as ZNFN3A1. In particular, the protein catalyzes the
methylation of histone H3 or a fragment of histone H3 comprising lysine
4. Whether a subject protein has the target activity can be determined by
the present invention. Namely, the methyl transferase activity can be
determined by contacting a polypeptide with a substrate (e.g., histone H3
or fragment comprising lysine 4) and a co-factor (e.g.
S-adenosyl-L-methionine) under conditions suitable for methylation of the
substrate and detecting the methylation level of the substrate.

[0043]Methods for preparing proteins functional equivalent to a given
protein are well known by a person skilled in the art and include known
methods of introducing mutations into the protein. For example, one
skilled in the art can prepare proteins functional equivalent to the
human ZNFN3A1 protein by introducing an appropriate mutation in the amino
acid sequence of the human ZNFN3A1 protein by site-directed mutagenesis
(Hashimoto-Gotoh, T. et al. (1995), Gene 152, 271-275; Zoller, M J, and
Smith, M. (1983), Methods Enzymol. 100, 468-500; Kramer, W. et al.
(1984), Nucleic Acids Res. 12, 9441-9456; Kramer W, and Fritz H J. (1987)
Methods. Enzymol. 154, 350-367; Kunkel, T A (1985), Proc. Natl. Acad.
Sci. USA. 82, 488-492; Kunkel (1988), Methods Enzymol. 85, 2763-2766).
Amino acid mutations can occur in nature, too. The protein used in the
present invention includes those proteins having the amino acid sequences
of the human ZNFN3A1 protein in which one or more amino acids are
mutated, provided the resulting mutated proteins are functional
equivalent to the human ZNFN3A1 protein. The number of amino acids to be
mutated in such a mutant is generally 10 amino acids or less, preferably
6 amino acids or less, and more preferably 3 amino acids or less. The
SET-domain "NHSCXXN" (SEQ ID NO:54) and "GEELXXXY" (SEQ ID NO:55) may be
conserved in the amino acid sequence of the mutated proteins for maintain
the methyl transferase activity ("X" indicates any amino acid).

[0046]An example of a protein to which one or more amino acids residues
are added to the amino acid sequence of human ZNFN3A1 protein (SEQ ID NO:
51) is a fusion protein containing the human ZNFN3A1 protein. Fusion
proteins are, fusions of the human ZNFN3A1 protein and other peptides or
proteins, and are used in the present invention. Fusion proteins can be
made by techniques well known to a person skilled in the art, such as by
linking the DNA encoding the human ZNFN3A1 protein of the invention with
DNA encoding other peptides or proteins, so that the frames match,
inserting the fusion DNA into an expression vector and expressing it in a
host. There is no restriction as to the peptides or proteins fused to the
protein of the present invention.

[0048]Fusion proteins can be prepared by fusing commercially available
DNA, encoding the fusion peptides or proteins discussed above, with the
DNA encoding the protein of the present invention and expressing the
fused DNA prepared.

[0049]An alternative method known in the art to isolate functional
equivalent proteins is, for example, the method using a hybridization
technique (Sambrook, J. et al., Molecular Cloning 2nd ed. 9.47-9.58, Cold
Spring Harbor Lab. Press, 1989). One skilled in the art can readily
isolate a DNA having high homology with a whole or part of the ZNFN3A1
DNA sequence (e.g., SEQ ID NO: 50) encoding the human ZNFN3A1 protein,
and isolate functional equivalent proteins to the human ZNFN3A1 protein
from the isolated DNA. The proteins used for the present invention
include those that are encoded by DNA that hybridize with a whole or part
of the DNA sequence encoding the human ZNFN3A1 protein and are functional
equivalent to the human ZNFN3A1 protein. These proteins include mammal
homologues corresponding to the protein derived from human or mouse (for
example, a protein encoded by a monkey, rat, rabbit and bovine gene). In
isolating a cDNA highly homologous to the DNA encoding the human ZNFN3A1
protein from animals, it is particularly preferable to use tissues from
skeletal muscle, testis, HCC, or colorectal tumors.

[0050]The condition of hybridization for isolating a DNA encoding a
protein functional equivalent to the human ZNFN3A1 protein can be
routinely selected by a person skilled in the art. For example,
hybridization may be performed by conducting prehybridization at
68° C. for 30 min or longer using "Rapid-hyb buffer" (Amersham
LIFE SCIENCE), adding a labeled probe, and warming at 68° C. for 1
hour or longer. The following washing step can be conducted, for example,
in a low stringent condition. A low stringent condition is, for example,
42° C., 2×SSC, 0.1% SDS, or preferably 50° C.,
2×SSC, 0.1% SDS. More preferably, high stringent condition is used.
A high stringent condition is, for example, washing 3 times in
2×SSC, 0.01% SDS at room temperature for 20 min, then washing 3
times in 1×SSC, 0.1% SDS at 37° C. for 20 min, and washing
twice in 1×SSC, 0.1% SDS at 50° C. for 20 min. However,
several factors such as temperature and salt concentration can influence
the stringency of hybridization and one skilled in the art can suitably
select the factors to achieved the requisite stringency.

[0051]In place of hybridization, a gene amplification method, for example,
the polymerase chain reaction (PCR) method, can be utilized to isolate a
DNA encoding a protein functional equivalent to the human ZNFN3A1
protein, using a primer synthesized based on the sequence information of
the DNA (SEQ ID NO: 50) encoding the human ZNFN3A1 protein (SEQ ID NO:
51).

[0052]Proteins that are functional equivalent to the human ZNFN3A1 protein
encoded by the DNA isolated through the above hybridization techniques or
gene amplification techniques, normally have a high homology to the amino
acid sequence of the human ZNFN3A1 protein. "High homology" (also
referred to as "high identity") typically refers to the degree of
identity between two optimally aligned sequences (either polypeptide or
polynucleotide sequences). Typically, high homology or identity refers to
homology of 40% or higher, preferably 60% or higher, more preferably 80%
or higher, even more preferably 85%, 90%, 95%, 98%, 99%, or higher. The
degree of homology or identity between two polypeptide or polynucleotide
sequences can be determined by following the algorithm in "Wilbur, W. J.
and Lipman, D. J. Proc. Natl. Acad. Sci. USA (1983) 80, 726-730".

[0053]A protein useful in the context of the present invention may have
variations in amino acid sequence, molecular weight, isoelectric point,
the presence or absence of sugar chains, or form, depending on the cell
or host used to produce it or the purification method utilized.
Nevertheless, so long as it has a function equivalent to that of a human
ZNFN3A1 protein (SEQ ID NO: 51), it is useful in the present invention.

[0054]The proteins useful in the context of the present invention can be
prepared as recombinant proteins or natural proteins, by methods well
known to those skilled in the art A recombinant protein can be prepared
by inserting a DNA, which encodes the protein of the present invention
(for example, the DNA comprising the nucleotide sequence of SEQ ID NO:
50), into an appropriate expression vector, introducing the vector into
an appropriate host cell, obtaining the extract, and purifying the
protein by subjecting the extract to chromatography, for example, ion
exchange chromatography, reverse phase chromatography, gel filtration, or
affinity chromatography utilizing a column to which antibodies against
the protein of the present invention is fixed, or by combining more than
one of aforementioned columns.

[0055]Also when the protein useful in the context of the present invention
is expressed within host cells (for example, animal cells and E. coli) as
a fusion protein with glutathione-S-transferase protein or as a
recombinant protein supplemented with multiple histidines, the expressed
recombinant protein can be purified using a glutathione column or nickel
column.

[0056]After purifying the fusion protein, it is also possible to exclude
regions other than the objective protein by cutting with thrombin or
factor-Xa as required.

[0057]A natural protein can be isolated by methods known to a person
skilled in the art, for example, by contacting the affinity column, in
which antibodies binding to the ZNFN3A1 protein described below are
bound, with the extract of tissues or cells expressing the protein of the
present invention. The antibodies can be polyclonal antibodies or
monoclonal antibodies.

[0058]In the present invention, methyl transferase activity of a ZNFN3A1
polypeptide can be determined by methods known in the art. For example,
the ZNFN3A1 and a substrate can be incubated with a labeled methyl donor,
under suitable assay conditions. A histone H3, histone H3 peptide, and
S-adenosyl-[methyl-14C]-L-methionine, or
S-adenosyl-[methyl-3H]-L-methionine preferably can be used as the
substrate and methyl donor, respectively. Transfer of the radiolabel to
the histone or histone peptide can be detected, for example, by SDS-PAGE
electrophoresis and fluorography. Alternatively, following the reaction
the histone or histone peptides can be separated from the methyl donor by
filtration, and the amount of radiolabel retained on the filter
quantitated by scintillation counting. Other suitable labels that can be
attached to methyl donors, such as chromogenic and fluorescent labels,
and methods of detecting transfer of these labels to histones and histone
peptides, are known in the art.

[0059]Alternatively, methyl transferase activity of ZNFN3A1 can be
determined using an unlabeled methyl donor (e.g. S-adenosyl-L-methionine)
and reagents that selectively recognize methylated histones or histone
peptides. For example, after incubation of the ZNFN3A1, substrate to be
methylated and methyl donor, under the condition capable of methylation
of the substrate, methylated substrate can be detected by immunological
method. Any immunological techniques using an antibody recognizing
methylated substrate can be used for the detection. For example, an
antibody against methylated histone is commercial available (abcam Ltd.).
ELISA or Immunoblotting with antibodies recognizing methylated histone
can be used for the present invention.

[0060]Instead of using antibodies, methylated histones can be detected
using reagents that selectively bind methylated histones with high
affinity. Such reagents are known in the art or can be determined by
screening assays known in the art. An exemplary binding reagent is
heterochromatin protein HP1, which binds histone H3 when methylated at
lysine 4 (H3-K4). HP1, or a binding fragment thereof, can be labeled, and
the HP1 or fragment bound to methylated H3-K4 detected. Alternatively,
the HP1 or fragment need not be labeled, and can instead be detected
using an anti-HP1 antibody in an ELISA assay.

[0061]In the present invention, an agent enhancing the methylation of the
substance can be used. For example, H3 methyltransferase activity of
Flag-tagged ZNFN3A1 was significantly higher in the presence of
S-adenosyl homocysteine hydrolase (SAHH) than the absence of SAHH (FIG.
3d). Thus, SAHH or functional equivalent thereof are preferable enhancing
agent for the methylation. The agent enhances the methylation of the
substance, the methyltransferase activity can be determined with higher
sensitivity thereby. ZNFN3A1 may be contacted with substrate and cofactor
under the existence of the enhancing agent.

[0062]Various low-throughput and high-throughput enzyme assay formats are
known in the art and can be readily adapted for detection or measuring of
methyl transferase activity of ZNFN3A1. For high-throughput assays, the
histone or histone peptide substrate can conveniently be immobilized on a
solid support, such as a multiwell plate, slide or chip. Following the
reaction, the methylated product can be detected on the solid support by
the methods described above. Alternatively, the methyl transferase
reaction can take place in solution, after which the histone or histone
peptide can be immobilized on a solid support, and the methylated product
detected. To facilitate such assays, the solid support can be coated with
streptavidin and the histone labeled with biotin, or the solid support
can be coated with anti-histone antibodies. The skilled person can
determine suitable assay formats depending on the desired throughput
capacity of the screen.

[0063]ZNFN3A1 or the functional equivalent requires heat shock protein 90A
(HSP90A) for expressing the methyl transferase activity. Therefore, a
compound that interferes binding between ZNFN3A1 or the functional
equivalent and HSP90A is useful for modulation of the methyl transferase
activity. The compound can be screened through the following method.
Thus, the present invention also provides a method of screening for a
compound for treating colorectal cancer or hepatocellular carcinoma, said
method comprising the steps of: [0064]a. contacting a polypeptide
selected from the group consisting of: [0065]i. a polypeptide comprising
the amino acid sequence of SEQ ID NO: 51; [0066]ii. a polypeptide
comprising the amino acid sequence of SEQ ID NO: 51 wherein one or more
amino acids are substituted, deleted, or inserted and said polypeptide
has a biological activity equivalent to the polypeptide consisting of the
amino acid sequence of SEQ ID NO: 51; [0067]iii. a polypeptide that
comprises the amino acid sequence having at least about 80% homology to
SEQ ID NO: 51; and [0068]iv. a polypeptide encoded by a polynucleotide
that hybridizes under stringent conditions to a polynucleotide consisting
of the nucleotide sequence of SEQ ID NO: 50, wherein the polypeptide has
a biological activity equivalent to a polypeptide consisting of the amino
acid sequence of SEQ ID NO: 51; with a heat shock protein 90A
polypepetide (HSP90A) in the presence of a test compound; [0069]b.
detecting binding between the polypeptide and HSP90A; [0070]c. comparing
the binding of the polypeptide and HSP90A in the presence of the test
compound with that in the absence of the test compound, and [0071]d.
selecting a test compound which decreases the binding of the polypeptide
and HSP90A.

[0072]In the present invention, the binding of the polypeptide and HSP90A
can be detected via any suitable method known to those of skill in the
art. For example, either of the polypeptide and HSP90A can be bound to
solid support, and the other can be labeled with labeling substances for
detection. Labeling substances such as radioisotope (e.g., 3H,
14C, 32P, 33P, 35S, 125I, 131I), enzymes
(e.g., alkaline phosphatase, horseradish peroxidase,
β-galactosidase, β-glucosidase), fluorescent substances (e.g.,
fluorescein isothiosyanete (FITC), rhodamine), and biotin/avidin, may be
used for the labeling of a polypeptide or HSP90A in the present method.
Methods for detection of the labeling substances are well known.

[0073]The present invention also encompasses the use of partial peptides
of a protein of the present invention. A partial peptide has an amino
acid sequence specific to the protein of the ZNFN3A1 and consists of less
than about 400 amino acids, usually less than about 200 and often less
than about 100 amino acids, and at least about 7 amino acids, preferably
about 8 amino acids or more, and more preferably about 9 amino acids or
more. The partial peptide can be used, for example, for the screening for
a compound that binds to the protein of the ZNFN3A1, and screening for
inhibitors of the binding between ZNFN3A1 and co-factor thereof such as
SAM. The partial peptide containing the SET-domain preferably used for
these screening.

[0074]A partial peptide used for the invention can be produced by genetic
engineering, by known methods of peptide synthesis, or by digesting the
protein of the invention with an appropriate peptidase. For peptide
synthesis, for example, solid phase synthesis or liquid phase synthesis
may be used.

[0076]In a further embodiment of the method for screening a compound for
treating or preventing HCC or colorectal cancer of the present invention,
the method utilizes the binding ability of ZNFN3A1 to co-factor thereof,
such as SAM. The proteins having a mutation in the SET-domain which binds
to S-adenosyl-L-methyonine inhibits the cell proliferation of cancer.
These findings suggest that the ZNFN3A1 exerts the function of cell
proliferation via its binding to molecules, such as
S-adenosyl-L-methyonine. Thus, the inhibition of the binding between the
ZNFN3A1 and the co-factor thereof leads to the suppression of cell
proliferation, and compounds inhibiting the binding serve as
pharmaceuticals for treating or preventing a HCC or colorectal cancer.

[0077]This screening method includes the steps of: [0078]a. contacting a
polypeptide comprising an a contiguous amino acid sequence from the amino
acid sequence of SEQ ID NO: 51, and wherein the amino acid sequence
comprises either or both of NHSCDPN (SEQ ID NO:52) and GEELTICY (SEQ ID
NO:53), with an S-adenosyl-L-methyonine in the presence of a test
compound; [0079]b. detecting binding between the polypeptide and
S-adenosyl-L-methyonine; [0080]c. comparing the binding of the
polypeptide and S-adenosyl-L-methyonine in the presence of the test
compound with that in the absence of the test compound, and [0081]d.
selecting a test compound which decreases the binding of the polypeptide
and S-adenosyl-L-methyonine.

[0082]The polypeptide to be used for the screening may be a recombinant
polypeptide or a protein derived from the nature, or may also be a
partial peptide thereof so long as it retains the binding ability to
S-adenosyl-L-methyonine. The polypeptide to be used in the screening can
be, for example, a purified polypeptide, a soluble protein, a form bound
to a carrier, or a fusion protein fused with other polypeptides.

[0084]Test compounds useful in the assays described herein can also be
antibodies that specifically bind ZNFN3A1 or partial ZNFN3A1 peptides
that lack methyl transferase activity. For example, antibodies (e.g.,
monoclonal antibodies) can be tested for the ability to block the binding
between ZNFN3A1 and its substrate, S-adenosyl-L-methyonine or HSP90A.
Similarly partial ZNFN3A1 peptides can be tested for the ability to
inhibit the binding between ZNFN3A1 and its substrate,
S-adenosyl-L-methyonine or HSP90A can be used as inhibitors of ZNFN3A1
activity. Such antibodies and partial peptides can thus be used as
inhibitors of ZNFN3A1 activity.

[0085]As a method of screening for compounds that inhibit the binding
between the ZNFN3A1 and S-adenosyl-L-methyonine, many methods well known
by one skilled in the art can be used. Such a screening can be carried
out as an in vitro assay system, for example, in a cellular system. More
specifically, first, either the polypeptide, or S-adenosyl-L-methyonine
is bound to a support, and the other member is added together with a test
sample thereto. Next, the mixture is incubated, washed, and the other
member bound to the support is detected and/or measured.

[0086]Examples of supports that may be used for binding proteins include
insoluble polysaccharides, such as agarose, cellulose, and dextran; and
synthetic resins, such as polyacrylamide, polystyrene, and silicon;
preferably commercial available beads and plates (e.g., multi-well
plates, biosensor chip, etc.) prepared from the above materials may be
used. When using beads, they may be filled into a column.

[0087]The binding of a polypeptide or S-adenosyl-L-methyonine to a support
may be conducted according to routine methods, such as chemical bonding,
and physical adsorption. Alternatively, a polypeptide may be bound to a
support via antibodies specifically recognizing the polypeptide.
Moreover, binding of a polypeptide to a support can be also conducted by
means of avidin and biotin binding.

[0088]The binding between polypeptide and S-adenosyl-L-methyonine is
carried out in buffer, for example, but are not limited to, phosphate
buffer and Tris buffer, as long as the buffer does not inhibit the
binding between the proteins.

[0089]In the present invention, a biosensor using the surface plasmon
resonance phenomenon may be used as a mean for detecting or quantifying
the binding the polypeptide and S-adenosyl-L-methyonine. When such a
biosensor is used, the interaction between the polypeptide and
S-adenosyl-L-methyonine can be observed real-time as a surface plasmon
resonance signal, using only a minute amount of polypeptide and without
labeling (for example, BIAcore, Pharmacia). Therefore, it is possible to
evaluate the binding between the polypeptide and polypeptide and
S-adenosyl-L-methyonine using a biosensor such as BIAcore.

[0090]Alternatively, either the polypeptide or polypeptide and
S-adenosyl-L-methyonine, may be labeled, and the label may be used to
detect or measure the bound polypeptide or polypeptide and
S-adenosyl-L-methyonine. Specifically, after pre-labeling one of the
polypeptide or S-adenosyl-L-methyonine, the labeled member is contacted
with the other member in the presence of a test compound, and then, bound
member are detected or measured according to the label after washing.

[0091]Labeling substances such as radioisotope (e.g., 3H, 14C,
32P, 33P, 35S, 125I, 131I), enzymes (e.g.,
alkaline phosphatase, horseradish peroxidase, β-galactosidase,
β-glucosidase), fluorescent substances (e.g., fluorescein
isothiosyanete (FITC), rhodamine), and biotin/avidin, may be used for the
labeling of a polypeptide or S-adenosyl-L-methyonine in the present
method. When the polypeptide or S-adenosyl-L-methyonine is labeled with
radioisotope, the detection or measurement can be carried out by liquid
scintillation. Alternatively, polypeptide or S-adenosyl-L-methyonine
labeled with enzymes can be detected or measured by adding a substrate of
the enzyme to detect the enzymatic change of the substrate, such as
generation of color, with absorptiometer. Further, in case where a
fluorescent substance is used as the label, the bound member may be
detected or measured using fluorophotometer.

[0092]Furthermore, the binding of the polypeptide and
S-adenosyl-L-methyonine can be also detected or measured using antibodies
to the polypeptide. For example, after contacting the
S-adenosyl-L-methyonine immobilized on a support with a test compound and
the polypeptide, the mixture is incubated and washed, and detection or
measurement can be conducted using an antibody against the polypeptide.

[0093]In case of using an antibody in the present screening, the antibody
is preferably labeled with one of the labeling substances mentioned
above, and detected or measured based on the labeling substance.
Furthermore, the antibody bound to the protein in the screening of the
present invention may be detected or measured using protein G or protein
A column.

[0094]The compound isolated by the screening is a candidate for drugs that
inhibit the methyl transferase activity of ZNFN3A1 and can be applied to
the treatment or prevention of HCC or colorectal cancer.

[0095]Moreover, compounds in which a part of the structure of the compound
inhibiting the methyl transferase activity of ZNFN3A1 is converted by
addition, deletion and/or replacement are also included in the compounds
obtainable by the screening method of the present invention.

[0096]As noted above, the compounds that inhibit the methyl transferase
activity of ZNFN3A1 can be either partial peptides that lack the methyl
transferase activity of ZNFN3A1 or can be antibodies against ZNFN3A1. As
used herein, the term "antibody" refers to an immunoglobulin molecule
having a specific structure, that interacts (i.e., binds) only with the
antigen that was used for synthesizing the antibody or with an antigen
closely related thereto. Furthermore, an antibody may be a fragment of an
antibody or a modified antibody, so long as it binds to the proteins
encoded by ZNFN3A1 gene. For instance, the antibody fragment may be Fab,
F(ab')2, Fv, or single chain Fv (scFv), in which Fv fragments from H
and L chains are ligated by an appropriate linker (Huston J. S. et al.
Proc. Natl. Acad. Sci. U.S.A. 85:5879-5883 (1988)). More specifically, an
antibody fragment may be generated by treating an antibody with an
enzyme, such as papain or pepsin. Alternatively, a gene encoding the
antibody fragment may be constructed, inserted into an expression vector,
and expressed in an appropriate host cell (see, for example, Co M S. et
al. J. Immunol. 152:2968-2976 (1994); Better M. and Horwitz A. H. Methods
Enzymol. 178:476-496 (1989); Pluckthun A. and Skerra A. Methods Enzymol.
178:497-515 (1989); Lamoyi E. Methods Enzymol. 121:652-663 (1986);
Rousseaux J. et al. Methods Enzymol. 121:663-669 (1986); Bird R. E. and
Walker B. W. Trends Biotechnol. 9:132-137 (1991)).

[0097]An antibody may be modified by conjugation with a variety of
molecules, such as polyethylene glycol (PEG). The present invention
provides such modified antibodies. The modified antibody can be obtained
by chemically modifying an antibody. Such modification methods are
conventional in the field. Alternatively, an antibody may comprise as a
chimeric antibody having a variable region derived from a nonhuman
antibody and a constant region derived from a human antibody, or a
humanized antibody, comprising a complementarity determining region (CDR)
derived from a nonhuman antibody, the frame work region (FR) derived from
a human antibody and the constant region. Such antibodies can be prepared
by using known technologies. Humanization can be performed by
substituting rodent CDRs or CDR sequences for the corresponding sequences
of a human antibody (see e.g., Verhoeyen et al., Science 239:1534-1536
(1988)). Accordingly, such humanized antibodies are chimeric antibodies,
wherein substantially less than an intact human variable domain has been
substituted by the corresponding sequence from a non-human species.

[0098]Fully human antibodies comprising human variable regions in addition
to human framework and constant regions can also be used. Such antibodies
can be produced using various techniques known in the art. For example in
vitro methods involve use of recombinant libraries of human antibody
fragments displayed on bacteriophage (e.g., Hoogenboom & Winter, J. Mol.
Biol. 227:381 (1991), Similarly, human antibodies can be made by
introducing of human immunoglobulin loci into transgenic animals, e.g.,
mice in which the endogenous immunoglobulin genes have been partially or
completely inactivated. This approach is described, e.g., in U.S. Pat.
Nos. 6,150,584, 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425;
5,661,016.

[0099]When administrating the compound isolated by the method of the
invention as a pharmaceutical for humans and other mammals, such as mice,
rats, guinea-pigs, rabbits, cats, dogs, sheep, pigs, cattle, monkeys,
baboons, and chimpanzees, the isolated compound can be directly
administered or can be formulated into a dosage form using known
pharmaceutical preparation methods. For example, according to the need,
the drugs can be taken orally, as sugar-coated tablets, capsules, elixirs
and microcapsules, or non-orally, in the form of injections of sterile
solutions or suspensions with water or any other pharmaceutically
acceptable liquid. For example, the compounds can be mixed with
pharmaceutically acceptable carriers or media, specifically, sterilized
water, physiological saline, plant-oils, emulsifiers, suspending agents,
surfactants, stabilizers, flavoring agents, excipients, vehicles,
preservatives, binders, and such, in a unit dose form required for
generally accepted drug implementation. The amount of active ingredients
in these preparations makes a suitable dosage within the indicated range
acquirable.

[0100]Examples of additives that can be mixed to tablets and capsules are,
binders such as gelatin, corn starch, tragacanth gum and arabic gum;
excipients such as crystalline cellulose; swelling agents such as corn
starch, gelatin and alginic acid; lubricants such as magnesium stearate;
sweeteners such as sucrose, lactose or saccharin; and flavoring agents
such as peppermint, Gaultheria adenothrix oil and cherry. When the
unit-dose form is a capsule, a liquid carrier, such as an oil, can also
be further included in the above ingredients. Sterile composites for
injections can be formulated following normal drug implementations using
vehicles such as distilled water used for injections.

[0101]Physiological saline, glucose, and other isotonic liquids including
adjuvants, such as D-sorbitol, D-mannnose, D-mannitol, and sodium
chloride, can be used as aqueous solutions for injections. These can be
used in conjunction with suitable solubilizers, such as alcohol,
specifically ethanol, polyalcohols such as propylene glycol and
polyethylene glycol, non-ionic surfactants, such as Polysorbate 80®
and HCO-50.

[0102]Sesame oil or Soy-bean oil can be used as a oleaginous liquid and
may be used in conjunction with benzyl benzoate or benzyl alcohol as a
solubilizer and may be formulated with a buffer, such as phosphate buffer
and sodium acetate buffer; a pain-killer, such as procaine hydrochloride;
a stabilizer, such as benzyl alcohol and phenol; and an anti-oxidant. The
prepared injection may be filled into a suitable ampule.

[0103]Methods well known to one skilled in the art may be used to
administer the pharmaceutical composition of the present invention to
patients, for example as intraarterial, intravenous, or percutaneous
injections and also as intranasal, transbronchial, intramuscular or oral
administrations. The dosage and method of administration vary according
to the body-weight and age of a patient and the administration method;
however, one skilled in the art can routinely select a suitable method of
administration. If said compound is encodable by a DNA, the DNA can be
inserted into a vector for gene therapy and the Vector administered to a
patient to perform the therapy. The dosage and method of administration
vary according to the body-weight, age, and symptoms of the patient but
one skilled in the art can suitably select them.

[0104]For example, although the dose of a compound that binds to the
ZNFN3A1 and regulates its activity depends on the symptoms, the dose is
about 0.1 mg to about 100 mg per day, preferably about 1.0 mg to about 50
mg per day and more preferably about 1.0 mg to about 20 mg per day, when
administered orally to a normal adult (weight 60 kg).

[0105]When administering parenterally, in the form of an injection to a
normal adult (weight 60 kg), although there are some differences
according to the patient, target organ, symptoms and method of
administration, it is convenient to intravenously inject a dose of about
0.01 mg to about 30 mg per day, preferably about 0.1 to about 20 mg per
day and more preferably about 0.1 to about 10 mg per day. Also, in the
case of other animals too, it is possible to administer an amount
converted to 60 kgs of body-weight.

[0106]The present invention further provides a method for treating a HCC
or colorectal cancer in a subject. Administration can be prophylactic or
therapeutic to a subject at risk of (or susceptible to) a disorder or
having a disorder associated with aberrant the methyl transferase
activity of ZNFN3A1. The method includes decreasing the function of
ZNFN3A1 in a HCC or colorectal cancer cell. Function can be inhibited
through the administration of a compound obtained by the screening method
of the present invention.

[0107]In another aspect the invention includes pharmaceutical, or
therapeutic, compositions containing one or more therapeutic compounds
described herein. Alternatively, the present invention also provides use
of one or more therapeutic compounds described herein for manufacturing a
pharmaceutical, or therapeutic, compositions for treating and/or
preventing of HCC or colorectal cancer. Pharmaceutical formulations may
include those suitable for oral, rectal, nasal, topical (including buccal
and sub-lingual), vaginal or parenteral (including intramuscular,
sub-cutaneous and intravenous) administration, or for administration by
inhalation or insufflation. The formulations may, where appropriate, be
conveniently presented in discrete dosage units and may be prepared by
any of the methods well known in the art of pharmacy. All such pharmacy
methods include the steps of bringing into association the active
compound with liquid carriers or finely divided solid carriers or both as
needed and then, if necessary, shaping the product into the desired
formulation.

[0108]Pharmaceutical formulations suitable for oral administration may
conveniently be presented as discrete units, such as capsules, cachets or
tablets, each containing a predetermined amount of the active ingredient;
as a powder or granules; or as a solution, a suspension or as an
emulsion. The active ingredient may also be presented as a bolus
electuary or paste, and be in a pure form, i.e., without a carrier.
Tablets and capsules for oral administration may contain conventional
excipients such as binding agents, fillers, lubricants, disintegrant or
wetting agents. A tablet may be made by compression or molding,
optionally with one or more formulational ingredients. Compressed tablets
may be prepared by compressing in a suitable machine the active
ingredients in a free-flowing form such as a powder or granules,
optionally mixed with a binder, lubricant, inert diluent, lubricating,
surface active or dispersing agent. Molded tablets may be made by molding
in a suitable machine a mixture of the powdered compound moistened with
an inert liquid diluent. The tablets may be coated according to methods
well known in the art. Oral fluid preparations may be in the form of, for
example, aqueous or oily suspensions, solutions, emulsions, syrups or
elixirs, or may be presented as a dry product for constitution with water
or other suitable vehicle before use. Such liquid preparations may
contain conventional additives such as suspending agents, emulsifying
agents, non-aqueous vehicles (which may include edible oils), or
preservatives. The tablets may optionally be formulated so as to provide
slow or controlled release of the active ingredient therein.

[0109]Formulations for parenteral administration include aqueous and
non-aqueous sterile injection solutions which may contain anti-oxidants,
buffers, bacteriostats and solutes which render the formulation isotonic
with the blood of the intended recipient; and aqueous and non-aqueous
sterile suspensions which may include suspending agents and thickening
agents. The formulations may be presented in unit dose or multi-dose
containers, for example sealed ampoules and vials, and may be stored in a
freeze-dried (lyophilized) condition requiring only the addition of the
sterile liquid carrier, for example, saline, water-for-injection,
immediately prior to use. Alternatively, the formulations may be
presented for continuous infusion. Extemporaneous injection solutions and
suspensions may be prepared from sterile powders, granules and tablets of
the kind previously described.

[0110]Formulations for rectal administration may be presented as a
suppository with the usual carriers such as cocoa butter or polyethylene
glycol. Formulations for topical administration in the mouth, for example
buccally or sublingually, include lozenges, comprising the active
ingredient in a flavored base such as sucrose and acacia or tragacanth,
and pastilles comprising the active ingredient in a base such as gelatin
and glycerin or sucrose and acacia. For intra-nasal administration the
compounds obtained by the invention may be used as a liquid spray or
dispersible powder or in the form of drops. Drops may be formulated with
an aqueous or non-aqueous base also comprising one or more dispersing
agents, solubilizing agents or suspending agents. Liquid sprays are
conveniently delivered from pressurized packs.

[0111]For administration by inhalation the compounds are conveniently
delivered from an insufflator, nebulizer, pressurized packs or other
convenient means of delivering an aerosol spray. Pressurized packs may
comprise a suitable propellant such as dichlorodifluoromethane,
trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or
other suitable gas. In the case of a pressurized aerosol, the dosage unit
may be determined by providing a valve to deliver a metered amount.

[0112]Alternatively, for administration by inhalation or insufflation, the
compounds may take the form of a dry powder composition, for example a
powder mix of the compound and a suitable powder base such as lactose or
starch. The powder composition may be presented in unit dosage form, in
for example, capsules, cartridges, gelatin or blister packs from which
the powder may be administered with the aid of an inhalator or
insufflators.

[0113]When desired, the above described formulations, adapted to give
sustained release of the active ingredient, may be employed. The
pharmaceutical compositions may also contain other active ingredients
such as antimicrobial agents, immunosuppressants or preservatives.

[0114]It should be understood that in addition to the ingredients
particularly mentioned above, the formulations of this invention may
include other agents conventional in the art having regard to the type of
formulation in question, for example, those suitable for oral
administration may include flavoring agents.

[0115]Preferred unit dosage formulations are those containing an effective
dose, as recited below, or an appropriate fraction thereof, of the active
ingredient.

[0116]For each of the aforementioned conditions, the compositions may be
administered orally or via injection at a dose of from about 0.1 to about
250 mg/kg per day. The dose range for adult humans is generally from
about 5 mg to about 17.5 g/day, preferably about 5 mg to about 10 g/day,
and most preferably about 100 mg to about 3 g/day. Tablets or other unit
dosage forms of presentation provided in discrete units may conveniently
contain an amount which is effective at such dosage or as a multiple of
the same, for instance, units containing about 5 mg to about 500 mg,
usually from about 100 mg to about 500 mg.

[0117]The pharmaceutical composition preferably is administered orally or
by injection (intravenous or subcutaneous), and the precise amount
administered to a subject will be the responsibility of the attendant
physician. However, the dose employed will depend upon a number of
factors, including the age and sex of the subject, the precise disorder
being treated, and its severity. Also the route of administration may
vary depending upon the condition and its severity.

[0120]The entire coding sequences of wild-type ZNFN3A1 and mutant ZNFN3A1
(ΔEEL and ΔNHSC) were cloned into the appropriate cloning
sites of p3xFLAG-CMV-10 (SIGMA). Plasmids designed to express the sense
strand of wild-type ZNFN3A1 (p3xFLAG-CMV-ZNFN3A1) or mutant ZNFN3A1
(p3xFLAG-CMV-ZNFN3A1-ΔEEL, p3xFLAG-CMV-ZNFN3A1-ΔNHSC), or
control plasmid (p3xFLAG-CMV-10), were transfected into HEK293,
colorectal cancer or hepatoma cells using FuGENE6 reagent according to
the supplier's recommendations (Roche). Transfected cells were maintained
in culture media supplemented with an optimized concentration of
geneticin. Cell viability was measured by Cell Counting Kit-8 according
to the manufacturer's protocol (DOJINDO).

Identification of Downstream Genes by cDNA Microarray.

[0121]HEK293 cells expressing no ZNFN3A1 were transfected with either
pcDNA-ZNFN3A1 or mock vector. RNA was extracted at 18 h after
transfection, labeled with Cy3 or Cy5 dye, and subjected to
co-hybridization onto in-house cDNA microarray slides containing 13,824
genes as described previously (2, 3). After normalization of the data,
genes with signals higher than the cut-off value were further analyzed.

Chromatin Immunoprecipitation (ChIP) Assays.

[0122]HEK293, HepG2 and Huh7cells were transfected with pFLAG-CMV-ZNFN3A1
and then fixed in 1% formaldehyde. The fixed chromatin samples were
subjected to immunoprecipitation using ChIP assay kit according to the
manufacturer's instructions (Promega). DNA from the HEK293 cells was
precipitated with anti-Flag antibody or anti-di-methylated histone H3
antibody, DNA from HepG2 or Huh7 cells with anti-ZNFN3A1 antibody. The
sets of primers used for ChIP assay were shown in Table 1.

[0123]The fragment of Nkx2.8 promoter were amplified by PCR using a set of
primer, 5'-AGCGGGCCTGGTACCAAATTTGTG-3' (SEQ ID NO;46) and
5'-CCGGGATGCTAGCGCATTTACAGC-3' (SEQ ID NO;47), and cloned the product
into pGL3 basic vector (pGL3-Nkx2.8-wtZBE). Mutant reporter plasmids
(pGL3-Nkx2.8-mutZBE) were prepared by replacing the ZNFN3 A1-binding
sequences (CCCTCCT to CCGACCT and GAGGGG to GTCGGG) in pGL3-Nkx2.8-wtZBE
using the QuickChange Site-Directed Mutagenesis Kit according to the
supplier's recommendations (Stratagene). Luciferase assays were carried
out using a Dual-Luciferase Reporter Assay System according to the
manufacturer's instructions (Promega).

Establishment of HEK293-Nkx2.8Luc Cells.

[0124]Stable transformant of HEK293-Nkx2.8Luc cells were established by
the transfection with pGL3-Nkx2.8-wtZBE and pcDNA(+)3.1 plasmids (10:1)
into HEK293 cells using FuGENE6 reagent according to the supplier's
recommendations (Roche). Transfected cells were maintained in culture
media supplemented with 0.9 μg/μl of geneticin, and single colonies
were selected two weeks after transfection.

Cell Lines

[0125]Human embryonic kidney 293 (HEK293) and human cervical cancer (HeLa)
cells were obtained from IWAKI. A human hepatoma cell line HepG2, a human
cervical cancer line HeLa, and a human colon cancer line HCT116 were
obtained from the American Type Culture Collection (ATCC). Another human
hepatoma cell line Huh7 was obtained from Japanese Collection of Research
Bioresources (JCRB), while SNU423 and SNU475 were obtained from the Korea
cell-line bank All cell lines were grown in monolayers in appropriate
media.

RT-PCR

[0126]Standard RT-PCR was carried out in a 20 μl volume of PCR buffer
(TAKARA), and amplified for 4 min at 94° C. for denaturing,
followed by 30 cycles of 94° C. for 30 s, 56° C. for 30 s,
72° C. for 30 s, in the Gene Amp PCR system 9700 (Perkin-Elmer).
Primer sequences used for the RT-PCR experiments were shown in table2.

[0128]Proteins containing the wild-type SET domain of ZNFN3A1 and two
forms of mutant protein that lacked one of the two regions were prepared,
and cross-linked equal amount of each protein with [3H]-labeled SAM
by exposure to UV-radiation. As shown in FIG. 1b, the wild-type SET
domain was capable of interacting with [3H]-labeled SAM and that
neither of the mutants interacted with it, indicating the interaction of
the SET domain with the methyl donor. The wild-type SET or control
recombinant protein of SET7 was further incubated with [3H]-labeled
SAM and a mixture of histones as substrates, and labeled protein after
the separation on SDS-PAGE was measured. As expected, a strong band
corresponding to [3H]-labeled histone H3 protein by the control SET7
(FIG. 1c, lane 3) was detected. When substrates were incubated with the
wild-type SET of ZNFN3A1, a faint band corresponding to labeled histone
H3 was detected, which was not observed with mock (FIG. 1c, lane 1 and
2).

[0129]Since yeast two-hybrid screening identified HSP90A as an interacting
protein of ZNFN3A1, it was hypothesized that HSP90A might assist the
protein folding of the SET. To test this hypothesis SAM and histones were
incubated in combination with the wild-type SET and recombinant HSP90A
protein, which resulted in enhanced methyl transferase activity onto
histone H3 (FIG. 1c, lane 4 and 5). These data demonstrate that ZNFN3A1
regulates expression of downstream genes through modification of
chromatin structure and the associated RNA polymerase II activity.

EXAMPLE 3

Histone H3 methyltransferase activity of ZNFN3A1

[0130]Since proteins containing SET domain play a crucial role in
methylation of histone H3 lysine 4 (H3-K4) or lysine 9 (H3-K9), we
investigated whether ZNFN3A1 has an ability to methylate H3-K4 or H3-K9.
We incubated recombinant histone H3 in the presence of SAM and HSP90A
with wild-type or mutant ZNFN3A1, or SET7 in vitro. In agreement with
previous reports (26), SET7 enhanced mono- and di-methylation of H3-K4,
but did not induce its tri-methylation (FIG. 2a, lane 2). On the other
hand, the wild-type ZNFN3A1 did not lead to mono-methylation. However, it
could cause di- and tri-methylation of H3-K4 (FIG. 2a, lane 3). This
methylation was completely inhibited by addition of di-methylated H3-K4
peptides, but was unaffected by that of di-methylated H3-K9 peptides
(FIG. 2b). The experiment using H3-K9 indicated that H3-K9 was methylated
by neither wild-type nor mutant ZNFN3A1 (FIG. 2c), suggesting that
ZNFN3A1 has the H3-K4-specific methyltransferase activity.

EXAMPLE 4

Histone H3-K4 methyltransferase activity of recombinant ZNFN3A1

[0131]In addition, the entire coding region of wild type or mutant ZNFN3A1
was cloned into an appropriate cloning site of pGEX6P-1 vector, and
expressed in the DH10B cells. Recombinant GST-ZNFN3A1 fusion protein was
purified with Sepharose 4B beads (Amersham), and recombinant ZNFN3A1 was
further separated using Precision protease (Amersham) according to the
supplier's protocols (FIG. 3a, lane 4). The recombinant ZNFN3A1 also
showed an HMTase activity to histone H3 in vitro (FIG. 3b). Western blot
analysis using anti-di-methylated and anti-tri-methylated H3-K4
antibodies confirmed that the recombinant ZNFN3A1 induced di-methylation
and tri-methylation on histone H3-K4 (FIG. 3c, upper and lower panel,
respectively). Since S-adenosyl homocysteine hydrolase (SAHH) hydrolyzes
SAH that is catalyzed from SAM and inhibits methylation, and enhances the
methyltransferase activity (4), we have investigated whether SAHH affect
on the histone H3 methyltransferase activity of ZNFN3A1. The H3
methyltransferase activity of Flag-tagged ZNFN3A1 was significantly
higher in the presence of SAHH than the absence of SAHH (FIG. 3d).

EXAMPLE 5

Association Between HMTase Activity of ZNFN3A1 and Proliferation of Cancer
Cells

[0132]To analyze the effect of HMTase activity on cell growth, we carried
out a colony-formation assay by transfecting plasmids expressing
wild-type (p3xFLAG-CMV-ZNFN3A1) or HMTase-inactive mutant forms
(p3xFLAG-CMV-ZNFN3A1-ΔEEL, p3xFLAG-CMV-ZNFN3A1-ΔNHSC) of
ZNFN3A1, or control plasmids (p3xFLAG-CMV). Transduction of wild-type
ZNFN3A1 produced markedly more colonies than control or mutant ZNFN3A1 in
HEK293 cells expressing no endogenous ZNFN3A1, which recapitulated
oncogenic activity of ZNFN3A1 (FIG. 4a). Consistently, transfection with
p3xFLAG-CMV-ZNFN3A1 increased the number of colonies compared to the
control in HCT116 colon cancer cells (FIG. 4b). On the other hand, that
with p3xFLAG-CMV-ZNFN3A1-ΔEEL reduced the growth of HCT116 cells
compared with p3xFLAG-CMV, suggesting that the mutant ZNFN3A1 may
interfere the function of endogeneous ZNFN3A1. Additionally, we also
investigated growth inhibitory effect of the mutant forms of plasmids in
various colorectal cancer cell lines and hepatoma cell lines (FIG. 5).
The result showed that transduction of HMTase-inactive ZNFN3A1
(p3xFLAG-CMV-ZNFN3A1-ΔEEL, p3xFLAG-CMV-ZNFN3A1-ΔHSC)
significantly reduced the growth of cancer cells compared to the control,
which may suggest that HMTase activity of ZNFN3A1 associates with
proliferation of colon cancer and hepatoma cells.

EXAMPLE 6

Identification of Genes Regulated by ZNFN3A1

[0133]To identify downstream genes regulated by ZNFN3A1, pcDNA-ZNFN3A1 was
transfected into HEK293 cells that showed undetectable level of ZNFN3A1
expression by RT-PCR, and monitored alterations in gene expression using
cDNA microarray containing 13,824 genes. Immunoblot analysis depicted
time-dependent induction of ZNFN3A1 as early as 12 hours (FIG. 6a),
therefore RNA was extracted from cells transfected with pcDNA-ZNFN3A1 and
those with pcDNA-mock at 18 hours after transfection. The expression
profile analysis identified 81 genes with altered expression including 62
genes that were up-regulated greater than three fold in pcDNA-ZNFN3A1
transfected cells compared with mock transfected cells, and 19 genes that
were down-regulated less than three fold (Table 5). Among the 62
up-regulated genes, a set of oncogenes such as Myc, Crk, JunD, Maf; and
Wnt10B, genes involved in cell cycle regulation (cyclin G1, Cdk2 and
Topoisomerase II), and homeobox genes (Nkx2.5, Nkx2.8 and LIM homeobox
protein 2) were found to be up-regulated by introduction of ZNFN3A1.
Associated with cyclinA and cyclinE, cdk2 plays an crucial role for
S-phase progression (5, 6), and the amplification of cyclinE/cdk2
complexes was shown to be involved in tumor progression in several tumors
including CRC and HCC (7, 8). It is well known that homeobox genes are
important factors for morphological change in development and for
tumorigenesis (9). Therefore elevated expression of ZNFN3A1 plays a
crucial role in human carcinogenesis through the activation of these
downstream genes.

[0134]11 up-regulated genes including Nkx2.8, C/EBPδ, Nkx2.5,
Wnt10B, PIK3CB, NEURL, PSMD9, ECEL1, CRKL, APS, and Seb4D were selected,
and semi-quantitative RT-PCR was performed using RNA from the cells
transfected with ZNFN3A1 or mock. The sets of primers used for
semi-quantitative RT-PCR were shown in Table 2.

[0135]Expectedly, the result corroborated enhanced expression of these
genes by ZNFN3A1 (FIG. 6b). The putative ZNFN3A1-binding sequences were
searched within 1.5-kb region upstream of transcription starting sites of
Nkx2.8. Two sequences were identified (FIG. 7a). Subsequent chromatin
immunoprecipitation (ChIP) assay using cells transfected with
pFLAG-ZNFN3A1 and anti-Flag M2 antibody confirmed that one genomic
segment (ChIP-4) containing these sequences associated with ZNFN3A1, and
that other segments (ChIP-1, -2, and -3) with no ZNFN3A1-binding
sequences did not (FIG. 7b). The ChIP-4 segment contained two putative
binding sequences (CCCTCCT and GAGGGG) within -510 bp to -467 bp of the
5' flanking region (FIG. 7a). Double stranded oligonucleotide probe was
prepared containing this ZNFN3A1-binding element (ZBE), and in vitro
binding assay was performed, using recombinant GST, GST-ZNFN3A1, and
GST-wtTcf4 as a control (FIG. 7c). The probe used for in vitro binding
assay are shown in Table 4.

[0136]The results indicate that oligonucleotide probe containing wild-type
Tcf4-binding motif (wtTBM) associated with GST-wtTcf4 but not with GST or
GST-ZNFN3A1 protein. Similarly, although ZBE did not associated with GST,
it was capable to bind with GST-ZNFN3A1 protein. Furthermore, it was
determined that the interaction was inhibited by the addition of cold
competitor DNA, suggesting specific interaction between GST-ZNFN3A1 and
ZBE.

[0137]To examine whether ZBE is responsible for transactivation of Nkx2.8
in cancer cells, a reporter plasmid was prepared that was cloned the -791
to +109 of Nkx2.8 including wild-type ZBE (pGL3-Nkx2.8-wtZBE) at an
upstream region of luciferase gene as well as that including mutant ZBE
(pGL3-Nkx2.8-mutZBE). These reporter plasmids were transfected into HepG2
or SNU475 cells, and their luciferase activity in the presence or absence
of siRNA to ZNFN3A1 was measured (FIG. 7d). The mutant reporter plasmid
revealed significantly lower activity than the wild-type plasmid in the
cells, indicating that ZBE is responsible for the transactivation of
Nkx2.8 in the cells. Notably, co-transfection with plasmids expressing
siRNA to ZNFN3A1 (psiU6BX-ZNFN3A1-12 were prepared by cloning the
following double stranded oligonucleotide into the Bbs1 site of the
psiU6BX vector; Forward:
5'-CACCAACATCTACCAGCTGAAGGTGTTCAAGAGACACCTTCAGCTGGTAGATGT T-3'(SEQ ID NO;
48), Reverse: 5'-AAAAAACATCTACCAGCTGAAGGTGTCTCTTGAACACCTTCAGCTGGTAGATGT
T-3'(SEQ ID NO; 49) (WO2004/76623)) reduced the luciferase activity of
wild-type reporter plasmids compared to mock (psiU6BX-Mock), but it did
not affect the activity of mutant plasmids.

[0138]These data indicate that ZNFN3A1 directly regulates transcriptional
activity of Nkx2.8 through the interaction to ZBE.

EXAMPLE 8

Nkx2.8 is Associated, with HSP90A-Dependent HMTase Activity of ZNFN3A1

[0139]To determine whether HMTase activity of ZNFN3A1 associates with the
expression of Nkx2.8, and whether HSP90A is involved in its regulation,
HEK293 cells were transfected with plasmids expressing wild-type or
HMTase-inactive mutant-ZNFN3A1 (ZNFN3A1-ΔEEL and
ZNFN3A1-ΔNHSC), and semi-quantitative RT-PCR was performed using
RNAs isolated from the transfected cells (FIG. 7e). As expected, although
wild-type plasmid enhanced the expression of Nkx2.8, both types of mutant
plasmids failed to induce the expression. Furthermore, addition of
geldanamycin, a specific inhibitor of HSP90A, suppressed the expression
enhancement caused by wild-type ZNFN3A1 (FIG. 7e lane 3), which is
consistent with the finding that HSP90A enhances HMTase activity of
ZNFN3A1 in vitro (FIG. 1c). HEK293-Nkx2.8Luc cells that integrated the
promoter region of Nkx2.8 and luciferase gene (pGL3-Nkx2.8-wtZBE) in the
genome were established. Transfection with plasmids expressing wild-type
ZNFN3A1 increased the luciferase activity in a dose-dependent manner,
whereas, that in addition of 2 μM geldanamycin or that with
HMTase-inactive mutant did not enhance the activity (FIG. 7f). Taken
together, these results indictate that expression of Nkx2.8 is associated
with HSP90A-dependent HMTase activity of ZNFN3A1.

EXAMPLE 9

Association Between ChaIP-4 Region in the Nkx2.8 Promoter and ZNFN3A1

[0140]We carried out an additional ChIP assay with anti-ZNFN3A1 antibody
using extracts from HepG2 or Huh7 hepatoma cells that abundantly
expressed ZNFN3A1, which corroborated an interaction between endogenous
ZNFN3A1 protein and the ChIP-4 region (FIG. 8a). Further ChIP assay with
anti-di-methylated H3-K4 antibody revealed an association between
di-methylated H3-K4 and the ChIP-4 region in HEK293 cells transfected
with wild-type ZNFN3A1 (FIG. 8b).

INDUSTRIAL APPLICABILITY

[0141]The present inventors have shown that ZNFN3A1 has methyl transferase
activity, and the suppression of the activity leads to inhibition of cell
proliferation of cancer cells. Thus, agents that inhibit the methyl
transferase activity or the binding of ZNFN3A1 and co-factor thereof
prevent its activity have therapeutic utility as anti-cancer agents,
particularly anti-cancer agents for the treatment of HCC or colorectal
cancer.

[0142]It has been reported that the expression of ZNFN3A1 is up-regulated
in HCC or colorectal cancer. Thus, the methods for detection of the
methyltransferase activity of ZNFN3A1 according to the present invention
is also useful for identification of these cancers. Specifically, cells
showing higher methyltransferase activity compared to normal cells can be
identified as cancer cells.

[0143]Furthermore, a modulator that regulates the methyltransferase
activity of ZNFN3A1 is also useful for identification of the cancers. For
example, such a modulator can be used to confirm whether the
methyltransferase activity detected in a subject cell is derived from
ZNFN3A1. Specifically, when the methyltransferase activity is modified
(inhibited or enhanced) by the modulator, the activity is judged as not
being false positive.

[0144]All patents, patent applications, and publications cited herein are
incorporated by reference in their entirety. Furthermore, while the
invention has been described in detail and with reference to specific
embodiments thereof, it will be apparent to one skilled in the art that
various changes and modifications can be made therein without departing
from the spirit and scope of the invention.